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Biomedical subjects

F A Siddiqui

Publications and source records attributed to F A Siddiqui.

At least 19 recordsLinked to original sources
PubMed2002

Hemoglobin binds melanoma cell tissue factor and enhances its procoagulant activity.

Tissue factor (TF), the membrane-bound glycoprotein that normally initiates the coagulation pathway, is expressed on the surface of various cells including endothelial cells, fibroblasts, monocytes and tumor cells. We recently reported that hemoglobin (Hb) enhances TF expression and procoagulant activity on TF-bearing human A375 malignant melanoma cells. To elucidate the mechanism of Hb-induced TF expression, we studied the interaction between purified TF from human A375 malignant melanoma cells and Hb. Selective binding of highly purified melanoma cell TF-apoprotein to Hb was demonstrated under native conditions using a dot-immunobinding assay and under denaturing conditions by Western blotting. The complex formation between purified melanoma cell TF-apoprotein and Hb was also demonstrated by the binding of fluid-phase Hb to immobilized TF-apoprotein (0-2.0 microg/ml) in an enzyme-linked immunosorbent assay. The binding was specific, concentration-dependent, saturable and inhibited significantly (60%) by Concanavalin-A. Hb enhanced the factor X-activating procoagulant activity of melanoma cell TF in a concentration-dependent manner, but had no effect on recombinant human TF. Concanavalin-A and wheat germ agglutinin significantly (60%) inhibited the Hb-induced procoagulant activity of malignant cell TF. We conclude that TF-apoprotein selectively binds Hb, most probably via the carbohydrate moieties (alpha-d-glucosyl; alpha-d-mannosyl and N-acetyl-beta-d-glucosaminyl residues) of TF, and enhances its procoagulant activity. The physiological significance of this interaction remains to be established.

Apoproteins↗
PubMed2001

Hemoglobin enhances tissue factor expression on human malignant cells.

Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.

Carcinoma, Transitional Cell↗
PubMed2001

Purification and properties of human melanoma cell tissue factor.

Tissue factor (TF) is a transmembrane glycoprotein that acts as a receptor for nonactivated and activated factor VII (FVII) and triggers the coagulation cascade. TF plays an important role in hemostasis, but may also have noncoagulation functions in vascular development, angiogenesis, and tumor cell metastasis. In tumor cells, analysis of the role of TF has been hampered by the lack of purified TF. In this study, TF antigen was identified on human A375 malignant melanoma cells using flow cytometry. We further purified TF apoprotein 2,000-fold to homogeneity from A375 melanoma cells using immunoaffinity chromatography. On SDS-polyacrylamide gel electrophoresis under reduction, purified TF apoprotein gave two major protein bands corresponding to molecular weights of 53 and 34 to 36 KD. The identity of these forms of TF was confirmed by Western blotting using a polyclonal antibody against human brain TF. Under reduction, the TF antibody bound with a monomeric form of TF (53 KD), and without reduction, to several forms of TF (34 to 128 KD). Preliminary carbohydrate analysis suggested that TF is a glycoprotein and contains about 22% total carbohydrates. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipids. The effects of varying concentrations (0 to 8 microg) of polyclonal antibodies to TF and FVII on TF procoagulant activity were studied. Both antibodies inhibited more than 70% of the procoagulant activity of TF in an FX activation assay. The complex formation between purified TF apoprotein and FVIIa was demonstrated by using an enzyme-linked immunosorbent assay. TF formed a complex with FVIIa in a concentration-dependent and saturable manner. We conclude that in human melanoma cells, TF occurs in monomeric and heterodimeric forms and appears to have similar properties as reported for TF from other sources.

Antibodies↗
PubMed1998

Pentoxifylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor.

Tissue factor (TF), the membrane glycoprotein that initiates blood coagulation, is constitutively expressed by many tumor cells and is implicated in peri-tumor fibrin deposition and hypercoagulability in cancer. Upregulation of tumor TF correlates with enhanced metastatic potential. Furthermore, TF has been colocalized with VEGF in breast cancer, specially at sites of early angiogenesis. There are no data on the effect of hypoxia on tumor cell TF expression. Since hypoxia is known to stimulate VEGF production, we studied whether this also induces tumor cell TF expression. Confluent monolayers of A375 melanoma, MCF-7 breast carcinoma and A549 lung carcinoma were cultured in either 95% air, 5% CO2 (normoxic) or 95% N2, 5% CO2 (hypoxic; 25-30 mmHg) for 24 h. Procoagulant activity (PCA) was measured by amidolytic and clotting assays, surface TF antigen by flow cytometry, early apoptosis by annexin V binding and VEGF levels in culture supernatants by ELISA. Hypoxia significantly increased tumor cell PCA in all three cell lines tested and TF antigen on A375 cells was increased four-fold (P <0.05). Pentoxifylline (PTX), a methylxanthine derivative, significantly inhibited the hypoxia-induced increase in PCA as well as VEGF release in all three cell lines tested. In A375 cells, PTX significantly inhibited TF antigen expression by both normoxic and hypoxic cells. Hypoxia induced a slight (5%) but not significant, increase in early apoptosis. Intravenous injection of hypoxic A375 cells into nude rats produced more pronounced thrombocytopenia (n = 5, P <0.01) and more lung metastases (n = 3, P <0.05) compared to normoxic cells. We conclude that hypoxia increases TF expression by malignant cells which enhances tumor cell-platelet binding and hematogenous metastasis. Hypoxia-induced upregulation of TF appears to parallel that of VEGF, although the mechanism remains unclear.

Animals↗
PubMed1996

Isolation, purification and properties of cathepsin B from buffalo liver.

Cathepsin B was isolated from buffalo liver by salt fractionation, ion-exchange resin treatment, gel filtration and repeated ion-exchange chromatography using a linear salt gradient. The enzyme showed activity, against denatured hemoglobin (or ovalbumin), alpha-N-benzoyl-DL-arginine p-nitroanilide (BAPNA), and alpha-benzoyl-DL-arginine-naphthylamine (BANA). It inactivated buffalo muscle aldolase with a half life period of 21 min. The pH-activity profiles obtained for the digestion of hemoglobin (or ovalbumin) and aldolase inactivation by the enzyme were found to be different. The enzyme (mol wt 27,800 by SDS-PAGE) eluted in gel filtration with a molecular weight of 27,000 and a Stokes radius of 2.31 nm. The results showed buffalo cathepsin B to be a single-chain molecule. The N- and C-terminal amino acids of the enzyme were found to be leucine and aspartic acid, respectively. It contained 0.7% concanavalin A reactive neutral carbohydrate. The amino acid composition of buffalo cathepsin B was found to be similar to that of human liver cathepsin B. The optical properties of the buffalo enzyme were found consistent with its aromatic amino acid content. The isoionic pH of the enzyme was found to be 5.70 and the intrinsic viscosity was 3.48 ml/g whence the frictional ratio, f/f0 was computed to be 1.10 suggesting that the native enzyme conformation is compact and is globular in solution.

Amino Acids↗
PubMed1993

Characterization of platelet agglutinating protein p37 purified from the plasma of a patient with thrombotic thrombocytopenic purpura.

We have previously reported the purification of a 37 KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura and have shown that it is present in a subset of TTP patients, but absent in normal subjects. In this study, we would like to report some of the physico-chemical and immunological properties of this protein. The native molecular weight of PAP p37 from gel filtration was found to be 36,000, which is in agreement with denatured molecular weight (37,000), determined by SDS--polyacrylamide gel electrophoresis under both reducing and non-reducing conditions. The values of Stoke's radius (25A), diffusion coefficient (8.59 x 10(-7)cm2/s) and frictional ratio (1.13), determined by molecular sieve chromatography, suggest that the native protein is compact and globular. The purified protein has an S20,w of 3.5s. Preliminary carbohydrate analysis suggested that p37 is a glycoprotein and contained about 11% neutral sugars and 6.6% sialic acid. Amino acid analysis indicated that the protein is relatively rich in aspartate and serine and has low cysteine, methionine and tryptophan contents. In dot immunobinding ELISA assay, PAP p37 did not react with antibodies to thrombospondin, fibrinogen, fibronectin, plasminogen and von Willebrand factor. Our results suggest that PAP p37 is a single polypeptide compact and globular glycoprotein and is immunologically not related to the aforementioned proteins.

Amino Acids↗
PubMed1992

Platelet-agglutinating protein p37 from a thrombotic thrombocytopenic purpura plasma forms complexes with platelet membrane glycoprotein IV (CD36).

We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAPp37) from the plasma of a patient with Thrombotic Thrombocytopenic purpura (TTP), and have shown recently that p37 causes platelet agglutination through its binding to membrane glycoprotein IV (GPIV). To gain further insight into the mechanism of p37 binding to GPIV, we have studied the interaction between p37 and GPIV. We now demonstrate specific complex formation of p37 with GPIV. In Western immunoblotting p37 binds to purified GPIV and the complex formed between the two proteins was detected by polyclonal antibody to p37 and peroxidase conjugated second antibody. No binding of p37 was noticed with the purified GPIIIa. A solid phase binding assay was developed to study the complex formation. Microtiter wells were coated with GPIV and the control proteins; 125I-p37 was added, allowed to bind and bound radioactivity was measured. Several lines of evidence indicate that the binding of p37 to GPIV was specific. a) GPIV immobilized on Immulon-2 wells bound 10-30 fold more p37 than immobilized fibrinogen, GPIIIa, and BSA. b) Polyclonal antibodies against p37 and GPIV inhibited the binding by 39-68% as compared with control IgG. c) GPIIIa antibody did not inhibit the binding. Molecular sieve chromatography of a mixture of 125I-p37 and GPIV also revealed the fluid phase complex formation ranging in molecular weight from 132,000 to over 350,000 daltons. These results show the specific interaction between p37 and GPIV and suggest that GPIV functions as a p37 receptor during platelet agglutination.

Antigens, CD↗
PubMed1991

Platelet agglutinating protein p37 causes platelet agglutination through its binding to membrane glycoprotein IV.

A 37 kDa platelet agglutinating protein (PAP p37) has previously been shown to be present in a subset of patients with thrombotic thrombocytopenic purpura and has been purified from their plasma. Using solubilized platelet membrane proteins from normal donors, it was shown by Western blotting that 125I-p37 bound to a membrane protein of 97 kDa (red/unred). Furthermore, the same protein was identified by reverse immunoblotting in which purified p37 was electrophoresed, transferred to the nitrocellulose sheet and incubated with solubilized normal platelet membrane proteins. The complex formed between p37 and the membrane protein was identified by autoradiography using polyclonal and monoclonal (OKM5) anti-GPIV antibodies, but was not detected by polyclonal antibody to GPIIIa. Similar studies with purified platelet GPIV under both reducing and non-reducing conditions demonstrated the binding of 125I-p37. Polyclonal and monoclonal antibodies to GPIV completely inhibited the platelet agglutination induced by TTP plasma containing p37, however, normal rabbit IgG, rabbit anti-GPIIIa IgG, and murine monoclonal anti-GPIIb/IIIa (10E5) antibodies had no effect. These data indicate that platelet GPIV is the receptor site for PAP p37.

Antibodies↗
PubMed1991

Binding of platelet agglutinating protein p37 from the plasma of a patient with thrombotic thrombocytopenic purpura to human platelets.

We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. To gain further insight into the interaction between PAP p37 and platelets, we have studied the properties of PAP p37 binding to platelets. Washed human platelets from two normal donors and two TTP patients after recovery were used for the experiments. The PAP p37 binding to platelets was specific, concentration dependent and saturable. Scatchard analysis demonstrated about 20,564-27,090 PAP p37 binding sites per platelet. Stimulation of platelets with thrombin or ADP did not have any significant effect on its binding. Thiol- and serine-specific protease inhibitors did not inhibit PAP p37 binding to the platelets. Sugars such as glucose, fructose, mannose, and sialic acid, at 40 mM, inhibited its binding to platelets by 44%, 73%, 79%, and 91% respectively, but galactose and amino sugars did not have any significant effect. At 250 micrograms/ml, Concanavalin-A inhibited 42% of binding, but other lectins, such as phytohemagglutinin-P, potato lectin and helix pomatia lectin (snail), did not. Pre-incubation of 125I-PAP p37 with the adult human IgG, decreased its binding to the platelets. The monoclonal antibodies to GP Ib (6D1) and GP IIb-IIIa complex (10E5) did not inhibit the binding of 125I-PAP p37 to platelets. Fibrinogen and von Willebrand factor did not affect the binding either. These results suggest that PAP p37 binds to platelets on the sites other than GP Ib or GP IIb-IIIa complex.

Antibodies, Monoclonal↗
PubMed1989

Electron microscopic study of platelet agglutination induced by thrombotic thrombocytopenic purpura plasma containing 37-KDa platelet agglutinating protein.

It has been demonstrated that plasma from a patient with thrombotic thrombocytopenic purpura (TTP) and 37-KDa platelet-agglutinating protein (PAP p37) purified from the same plasma caused the agglutination of platelets from normal subjects as well as from the same patient after recovery without the requirement of extracellular Ca++ and fibrinogen. Experiments were designed to study the morphologic changes of platelets as a result of agglutination and the distribution of platelet receptors for PAP p37 under transmission electron microscope. Following incubation with TTP plasma or PAP p37 with stirring, platelets showed shape change, pseudopod formation, variable degrees of degranulation, dilatation of open canalicular systems and formation of agglutinates composed of a few to several hundred platelets. After platelets were incubated with TTP plasma or PAP p37 they were washed and further incubated with rabbit anti-PAP p37 serum without stirring followed by immuno-staining. Abundant electron dense reaction products were bound directly and randomly to the outer surface of the membrane of solitary platelets. When the reaction mixture was stirred, electron dense particles were also present between the platelet membranes in the agglutinates. No staining was observed in control experiments using normal plasma or non-immune rabbit serum. These results indicate that the TTP plasma containing PAP p37 causes agglutination, shape change, and variable degrees degranulation in platelets and that PAP p37 binds randomly to the outer surface of platelet membrane.

Blood Platelets↗
PubMed1988

Platelet-agglutinating protein P37 from a thrombotic thrombocytopenic purpura plasma forms a complex with human immunoglobulin G.

We have previously reported the purification of a 37-kd platelet-agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. The platelet agglutination induced by PAP p37 has been shown to be inhibited by IgG from normal human adults and the same TTP patient after recovery. To elucidate the mechanism of inhibition of IgG, the interaction between PAP p37 and IgG was studied. The complex formation was demonstrated by the binding of fluid-phase IgG from normal adults and the same TTP patient after recovery to adsorbed PAP by using an enzyme-linked immunosorbent assay. The binding was specific, concentration dependent, and saturable. IgG purified from a 5-month-old baby and the same TTP patient during active disease did not form complex with PAP p37. The IgG covalently cross-linked to Sepharose 4B bound 125I-PAP p37 but not 125I-fibrinogen. Sucrose density gradient ultracentrifugation of a mixture of 125I-PAP p37 and IgG also revealed the fluid-phase complex formation with a sedimentation value of 19S. Complexes of molecular weight ranging from 180,000 to over 350,000 daltons were also detected by molecular sieve chromatography. The IgG that was bound to PAP p37 conjugated to Sepharose 4B inhibited the agglutination of washed platelets induced by TTP plasma containing PAP p37, whereas the IgG that was not bound to PAP p37 did not have a significant inhibitory effect. The complex formation between PAP p37 and specific IgG is likely to account for the in vitro inhibition of TTP plasma-induced agglutination and, at least partly, the in vivo successful treatment with specific IgG-containing normal plasma.

Blood Proteins↗
PubMed1985

Generation of multiple forms of methionyl-tRNA synthetase from the multi-enzyme complex of mammalian aminoacyl-tRNA synthetases by endogenous proteolysis.

Methionyl-tRNA synthetase occurs free and as high-molecular-weight multi-enzyme complexes in rat liver. The free form is purified to near homogeneity by conventional column chromatography and affinity chromatography on tRNA-Sepharose. The native molecular weight of free methionyl-tRNA synthetase is 64 500, based on its sedimentation coefficient of 4.5 S and Stokes radius of 33 A. The free methionyl-tRNA synthetase apparently belongs to alpha-type subunit structure, since the subunit molecular weight is 68 000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Methionyl-tRNA synthetase is dissociated from the high-molecular-weight synthetase complex by controlled trypsinization, according to Kellermann, O., Viel, C. and Waller, J.P. (Eur. J. Biochem. 88 (1978) 197-204). The dissociated, free methionyl-tRNA synthetase is subsequently purified to near homogeneity. The subunit structure of dissociated methionyl-tRNA synthetase is identical to that of endogenous free methionyl-tRNA synthetase. Anti-serum raised against Mr 104 000 protein in the synthetase complex, specifically inhibited methionyl-tRNA synthetase in both the free and the high-molecular-weight forms to the same extent. These results suggest that the occurrence of multiple forms of methionyl-tRNA synthetases in mammalian cells may, in part, be due to proteolytic cleavage.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Novel platelet-agglutinating protein from a thrombotic thrombocytopenic purpura plasma.

A novel platelet-agglutinating protein (PAP) was purified approximately 2,000-fold from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) by ammonium sulfate fractionation, DEAE-Sephacel and concanavalin A-Sepharose chromatographies. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with and without reduction, this preparation revealed a major protein band with a molecular weight of 37,000, and a minor band with a molecular weight of 32,000-34,000. After elution from the gel, only the 37,000-mol wt protein corresponding to the major band induced the platelet agglutination. When four normal plasmas and the recovery plasma from the same TTP patient were subjected to the similar purification steps, the 37,000-mol wt major band was absent. The 125I-PAP bound to the platelets in a concentration-dependent manner. The platelet agglutination induced by PAP was not inhibited by hirudin, heparin in the presence of antithrombin III, phenylmethylsulfonyl fluoride, apyrase, aspirin, or prostaglandin I2. However, it was inhibited by IgG from normal adults and from the same TTP patient after recovery. The anti-37,000-mol wt PAP antiserum prepared in the rabbit formed a single precipitin line against the highly purified PAP. Using this antiserum in the Western immunoblotting, the 37,000-mol wt protein band was found in the three TTP plasmas, of which the platelet-agglutinating activity was inhibited by the anti-37,000-mol wt PAP IgG. The 37,000-mol wt immunoprecipitin band was absent in the plasmas obtained from another two TTP patients, two normal subjects, two patients with idiopathic thrombocytopenic purpura, and two patients with disseminated intravascular coagulation. These results suggest that the 37,000-mol wt PAP is present only in certain cases of TTP, and is likely to be responsible for the formation of platelet thrombi in the microcirculation.

Adult↗
PubMed1985

Investigation of the role of von Willebrand factor in thrombotic thrombocytopenic purpura.

Von Willebrand factor (vWF) has been implicated to function as a cofactor in platelet aggregation induced by thrombotic thrombocytopenic purpura (TTP) plasma. To investigate further this role of vWF, we have used rabbit monospecific anti-FVIII/vWF antibodies and a monoclonal antibody to platelet glycoprotein Ib (GP Ib) that blocks the ristocetin-induced platelet aggregation. The monoclonal anti-platelet GP Ib antibody inhibited the platelet aggregation induced by ristocetin in the presence of normal plasma, but not that by any of the five TTP plasma samples. The TTP plasma samples from five patients were incubated with the monospecific antibodies to FVIII/vWF. In all of the samples, the FVIII/vWF:Ag was drastically reduced; however, there was almost no effect on the platelet-aggregating activity. Therefore, it is concluded that vWF is unlikely to play a major role in platelet aggregation induced by majority of TTP plasmas and that the site of platelet GP Ib, to which vWF binds in the presence of ristocetin, is not involved in TTP plasma-induced aggregation.

Antibodies, Monoclonal↗
PubMed1984

Purification and some properties of a protein obtained from normal human plasma which inhibits the platelet aggregation induced by thrombotic thrombocytopenic purpura plasma.

Plasma from patients with thrombotic thrombocytopenic purpura (TTP) caused the aggregation of washed human platelets, which was inhibited by preincubation with normal plasma. Using salt fractionation, ion exchange chromatography, and preparative agarose gel electrophoresis, we purified a protein from normal plasma which inhibited the platelet aggregation caused by TTP plasma. On SDS polyacrylamide gel, the purified inhibitor gave a single band with a M.W. of 150,000. The antiserum against the purified protein neutralized the activity of the inhibitor and formed an identical precipitin line against normal and TTP plasma.

Blood Coagulation Factors↗
PubMed1984

Inhibition of platelet-aggregating activity in thrombotic thrombocytopenic purpura plasma by normal adult immunoglobulin G.

Plasma from patients with thrombotic thrombocytopenic purpura (TTP) caused the aggregation of autologous and homologous platelets, and effect which was inhibited by normal plasma. IgG purified from seven normal adults at a concentration of 0.7 mg/ml completely inhibited the platelet aggregation induced by plasma obtained from two TTP patients with active disease. The inhibition of platelet aggregation by human adult IgG was concentration dependent, and the inhibitory activity of human IgG was neutralized by rabbit antihuman IgG. Fab fragments inhibited the TTP plasma-induced platelet aggregation as well as intact IgG, whereas Fc fragments had no effect. Platelet aggregation caused by ADP, collagen, epinephrine, or thrombin was not affected by purified human IgG. The prior incubation of IgG with TTP plasma caused a significantly greater reduction of platelet aggregation by TTP plasma than that of IgG and platelet suspension, suggesting that the IgG inhibits TTP plasma-induced platelet aggregation through direct interaction with platelet aggregating factor in TTP plasma. IgG obtained initially from five infants and young children under the age of 4 yr did not possess any inhibitory activity. When one of the children reached 3 yr of age, his IgG inhibited the aggregation induced by one TTP plasma, but not that caused by another plasma. The IgG procured from the same boy at 4 yr of age inhibited the aggregation induced by both TTP plasmas. The IgG purified from the TTP plasma during active disease failed to inhibit the aggregation caused by the same plasma. After recovery, however, the IgG effectively inhibited aggregation. These observations suggest that platelet-aggregating factors present in the TTP plasma are heterogeneous in nature and that the IgG present in the normal adult plasma, which inhibits the TTP plasma-induced platelet aggregation, may be partially responsible for the success of plasma infusion therapy in TTP.

Adult↗
PubMed1983

Purification and immunological characterization of DNA polymerase-alpha from human acute lymphoblastic leukemia cells.

DNA polymerase-alpha was purified from the cytosol of blast cells of a patient with acute lymphoblastic leukemia by ammonium sulfate fractionation and successive column chromatographies. The purified enzyme had a specific activity of 2943 units/mg protein with activated calf thymus DNA as a template. The enzyme sediments under high-salt conditions as a homogeneous band at 7.2 S and free from other DNA polymerases (beta, gamma) and terminal deoxynucleotidyl transferase activity. The native molecular weight of the enzyme from gel filtration and glycerol gradient centrifugation was found to be 175 000. The values of Stokes radius (53 A), diffusion coefficient (4.05 x 10(-7) cm2/s) and frictional ratio (1.42) determined by gel filtration suggest that the native enzyme is compact and globular. Antibodies to DNA polymerase-alpha were raised in rabbits. These antibodies, partially purified by 50% ammonium sulfate saturation and Sephadex G-200 chromatography, gave one precipitin band on immunodiffusion and inactivate DNA polymerase-alpha-. This antibody preparation also inhibited, in vitro, the activity of DNA polymerase-alpha from calf thymus, phytohemagglutinin-stimulated normal human lymphocytes, as well as that from other leukemic cells. Thus, DNA polymerase-alpha from calf thymus and human leukemic cells resemble each other in antibody specificity.

DNA Polymerase II↗